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Cyclohexenyl nucleic acids and altritol nucleic acids were evaluated as RNA modifications in an siRNA experiment targeting the P-glycoprotein expression. Both modified oligonucleotides are well accepted as RNA mimics leading to an increase in potency and duration of action of the gene-silencing effect.
Double-stranded RNA can induce the degradation of homologous RNAs in organisms as diverse as protozoa, animals, plants and fungi, resulting in post-transcriptional gene silencing. But in some species, RNA-mediated processes can also lead to translational repression, DNA methylation, heterochromatin formation or DNA elimination. In some situations, amplification of the 'trigger' double-stranded RNA seems to be required for efficient silencing. These findings imply that RNA-mediated mechanisms can control gene expression at both the transcriptional and the post-transcriptional level, and that they can operate in the nuclear and the cytoplasmic compartments.
Influenza A virus infection is a major source of morbidity and mortality worldwide. Because the effectiveness of existing vaccines and antiviral drugs is limited, development of new treatment modalities is needed. Here, we show that short interfering RNAs (siRNAs) specific for conserved regions of influenza virus genes can prevent and treat influenza virus infection in mice. Virus production in lungs of infected mice is reduced by siRNAs given either before or after initiating virus infection, by using slow i.v. administration of small volumes containing siRNAs in complexes with a polycation carrier. Similar effects also are observed when mice are given DNA vectors i.v. or intranasally, from which siRNA precursors can be transcribed. Development of delivery systems that may be compatible with human use demonstrates the potential utility of siRNAs for prophylaxis and therapy of influenza virus infections in humans.
AIM: To investigate the role of human La protein in HBV mRNA expression. METHODS: Three human La protein (hLa) specific siRNA expression cassettes (SECs) containing U6+1 promoter were prepared via one-step overlapping extension PCR. After transfection with SECs into HepG2 cells, inhibition effects on hLa expression were analyzed by semi-quantitative RT-PCR and Western blotting. Then, effective SECs were screened out and transfected into 2.2.15 cells, a stable HBV-producing cell line. HBV surface antigen (HBsAg) and e antigen (HBeAg) secretions into culture media were detected by microparticle enzyme immunoassay (MEIA) and HBs and HBe mRNA levels were analyzed by semi-quantitative RT-PCR. RESULTS: SEC products containing U6+1 snRNA promoter, and 3 sites of hLa mRNA specific siRNA were obtained successfully by one-step overlapping extension PCR and could be directly transfected into HepG2 cells, resulting in inhibition of La protein expression in both mRNA and protein levels, among which U6+1-hLa833 was the most efficient, which reduced 18.6-fold mRNA and 89% protein level respectively. In 2.2.15 cells, U6+1-hLa833 was also efficient on inhibition of hLa expression. Furthermore, semi-quantitative RT-PCR showed that HBs and HBe mRNA levels were significantly decreased by 8- and 66-fold in U6+1-hLa833 transfected cells compared to control. Accordingly, HBsAg and HBeAg secretions were decreased partly posttransfection with SECs. CONCLUSION: PCR-based SECs can be used to mediate RNAi in mammalian cells and provide a novel approach to study the function of La protein. The inhibition of La protein expression can result in a significant decrease of HBV mRNA, which implies that the hLa protein is also involved HBV RNA metabolism as one of the HBV RNA-stabilizing factors in human cells.
Pluripotent embryonic stem (ES) cells are an important model system to examine gene expression and lineage segregation during differentiation. One powerful approach to target and inhibit gene expression, RNAi, has been applied to ES cells with the goal of teasing out the cascades of gene expression/repression that shape the early embryo. In this chapter, we describe the current understanding of the mechanisms of gene silencing by small hairpin RNAs, as well as controls and caveats to using this approach in ES cells. A consideration of synthetic vs plasmid-based RNAi vectors, design of targeting constructs, transfection of ES cells, and flow sorting of targeted cells is followed by methods for the analysis of phenotype and behavior of targeted cell populations using immunohistochemistry, reverse transcriptase polymerase chain reaction, Western blotting, and scanning electron microscopy.
OBJECTIVE: To screen for siRNAs that inhibit the expression of p42(MAPK) in HeLa cell line. METHODS: Three p42(MAPK) siRNAs and one random siRNA were synthesized using Silencer siRNA Construction Kit, and labeled with Cy-3 for measurement of transfection effect. SiRNAs were transfected into HeLa cells by Lipofectamin 2000. The expression of p42(MAPK) was analyzed by Western blot. The biological effect of siRNAs on HeLa cell growth was monitored by MTT and flow cytometry. RESULTS: Two siRNAs (siRNA-2 and siRNA-3) among three tested were identified to be able to downregulate the p42(MAPK) expression. A concurrent growth retardation of HeLa cell line was observed in comparison with the control. CONCLUSION: Inhibition of p42(MAPK) expression with siRNA technique can inhibit the proliferation of HeLa cells.
Hec1 (highly expressed in cancer) plays an important role in chromosome segregation by interacting with a subset of checkpoint proteins that survey proper chromosome alignment and bipolar spindle attachment. In order to disrupt mitotic progression of tumor cell lines, we have used retrovirus and adenovirus vectors that inhibit Hec1 synthesis. Vector-expressed short hairpin RNAs (shRNAs) caused very efficient depletion of the target protein, cellular arrest and considerable mitotic catastrophe induction 96 h post infection in human cervix-adenocarcinoma (HeLa) and glioblastoma (U-373-MG) cell lines. Furthermore, adenocarcinomas induced in the flanks of nude mice show significant reduction in size compared with control when treated with either Hec1-shRNA retroviruses or adenoviruses. These results indicate that depletion of Hec1 could be used as a new strategy to block the dividing cell, and therefore against cancer.
Angiotensin II exerts its physiological effects by activating multiple subtypes of its receptor such as AT1a-, AT1b-, and AT2-receptors. Because of a high degree of similarity among these G-protein-coupled receptors, it has been difficult to assign diverse physiological actions of angiotensin II through these receptor subtypes. We have developed small interfering RNAs to selectively inhibit the expression of the AT1a receptor (AT1aR) subtype. A dsRNA, AT1 47, was found to be highly selective and efficient in reducing the levels of AT1aR subtype. Transfection of AT1aR-expressing CHO cells with dsRNA AT1 47 resulted in an 80% decrease in the AT1aR expression. In contrast, dsRNA AT1 47 showed no significant effects on both AT1bR and AT2R subtypes. Thus, AT1 47 provides us with a powerful tool to selectively silence this subtype of receptor to investigate its role in cardiovascular physiology.
Xenotransplantation from pigs to human beings is viewed as a potential solution for the acute organ shortage. However, consequent xenorejection induced by Gal alpha 1,3 Gal (a Gal, Gal antigen) prevents xenotransplantation from clinical application. Thus, the most attracting attempt to prevent xenorejection is the elimination of Gal. Our study suggested that compared with the human alpha 1,2 fucosyltransferase (FT) gene and the porcine antisense alpha 1,3 galactosyltransferase gene, sequence-specific siRNA targeting Gal was capable of suppressing Gal expression markedly, and therefore, significantly inhibiting xenoreactivity and the complement activation with human serum in PIEC cells. We also demonstrated the concordant inhibitory effect of siRNA and the human FT gene on Gal and corresponding functions, which implied a practical significance of combined transgenic strategy. The successful application of vector-based dsRNA-GT may extend the list of available modalities in the abrogation of xenorejection in xenotransplantation.
The crustacean hyperglycemic hormone (CHH) plays an important role in the regulation of hemolymph glucose levels, but it is also involved in other functions such as growth, molting and reproduction. In the present study we describe the first CHH family gene isolated from the Atlantic Ocean shrimp Litopenaeus schmitti. Sequence analysis of the amplified cDNA fragment revealed a high nucleotide sequence identity with other CHHs. Northern blot analysis showed that the isolated CHH mRNA from L. schmitti is present in the eyestalk but not in muscle or stomach. We also investigated the ability of dsRNA to inhibit the CHH function in shrimps in vivo. Injection of CHH dsRNA into the abdominal hemolymph sinuses resulted in undetectable CHH mRNA levels within 24 h and a corresponding decrease in hemolymph glucose levels, suggesting that functional gene silencing had occurred. These findings are the first evidence that dsRNA technique is operative in adult shrimps in vivo.
Delivery of siRNA to immune cells has been one of the major obstacles to widespread application of RNAi in the immunology field. Here, we report that osmotic delivery of siRNA can be used to silence genes in macrophage RAW264.7 without incurring either cytotoxic or immunomodulatory activity. We also showed usefulness of the osmotic delivery in other types of cells including T cell DO11.10. By repeated osmotic delivery of siRNA, long-term gene silencing was readily achieved. When TLR4 was disrupted in RAW264.7 cells for 48 h and the cells were stimulated with the TLR4 ligand LPS, a significant decrease in TNFalpha production was observed. DNA microarray-based gene expression profile analysis showed that gene silencing by osmotic delivery of siRNA was target-specific and the delivery method itself had little influence on overall gene expression.
Signal transduction through the IGF axis is implicated in proliferation, differentiation and survival during development and adult life. The IGF axis includes the IGF binding proteins (IGFBPs) that bind IGFs with high affinity and modulate their activity. In neuroblastoma (NB), a malignant childhood tumor, we found that IGFBP-5 is frequently expressed. Since NB is an IGF2-sensitive tumor, we investigated the relevance and the function of endogenous IGFBP-5 in LAN-5 and in SY5Y(N) cell lines transfected with micro and small interfering RNAs directed to IGFBP-5 mRNA. Cells in which IGFBP-5 expression was suppressed were growth-inhibited and more prone to apoptosis than the parental cell line and controls. Apoptosis was further enhanced by X-ray irradiation. The ability of these cells to undergo neuronal differentiation was impaired after IGFBP-5 inhibition but the effect was reversed by exposure to recombinant IGFBP-5. Together, these data demonstrate the importance of IGFBP-5 for NB cell functions and suggest that IGFBP-5 might serve as a novel therapeutic target in NB.
Short interfering RNAs (siRNAs), namely siUL54-1 and siU54-2 targeting UL54 (DNA polymerase) gene, and siUL97-1 and siUL97-2 targeting UL97 (phosphotransferase) gene, were used to inhibit respective genes of Human cytomegalovirus (HCMV) and consequently the virus infection process in human foreskin fibroblast (HFF) cultures. The virus infection was monitored by cell morphology (CPE), levels of UL83 and IE86 mRNAs, and virus antigen. The results showed that siUL97-2 remarkably inhibited viral CPE while other siRNAs were less inhibitory. The siRNAs reduced the levels of UL83 mRNA but not that of IE86 mRNA; again, siUL97-2 was most inhibitory. Particularly, siUL97-2 reduced the UL83 mRNA level 14, 19, 203, and 37 times at 24, 48, 72, and 96 hrs post infection (p.i.), respectively. When tested for the effect on viral antigen by immunofluorescent assay (IFA), UL97-2 exerted a marked inhibition. These results demonstrate the effectiveness of siRNAs against experimental HCMV infection and indicate their therapeutic potential.
Microtubule (MT)-based motor proteins, kinesins and dyneins, play important roles in multiple cellular processes including cell division. In this study, we describe the generation and use of an Escherichia coli RNase III-prepared human kinesin/dynein esiRNA library to systematically analyze the functions of all human kinesin/dynein MT motor proteins. Our results indicate that at least 12 kinesins are involved in mitosis and cytokinesis. Eg5 (a member of the kinesin-5 family), Kif2A (a member of the kinesin-13 family), and KifC1 (a member of the kinesin-14 family) are crucial for spindle formation; KifC1, MCAK (a member of the kinesin-13 family), CENP-E (a member of the kinesin-7 family), Kif14 (a member of the kinesin-3 family), Kif18 (a member of the kinesin-8 family), and Kid (a member of the kinesin-10 family) are required for chromosome congression and alignment; Kif4A and Kif4B (members of the kinesin-4 family) have roles in anaphase spindle dynamics; and Kif4A, Kif4B, MKLP1, and MKLP2 (members of the kinesin-6 family) are essential for cytokinesis. Using immunofluorescence analysis, time-lapse microscopy, and rescue experiments, we investigate the roles of these 12 kinesins in detail.
HT1080 cells stably expressing green fluorescent protein (GFP) linked to a 3' terminal AU-rich element (ARE) proved to be a convenient system to study the dynamics of mRNA stability, as changes in mRNA levels are reflected in increased or decreased fluorescence intensity. This study examined whether mRNA stability can be regulated by small interfering RNAs (siRNAs) targeted to AU-binding proteins (AUBPs), which in turn should reveal their intrinsic role as stabilizers or destabilizers of ARE-mRNAs. Indeed, siRNAs targeting HuR or BRF1 decreased or increased fluorescence, respectively. This effect was abolished if cells were treated with both siRNAs, thus indicating antagonistic control of ARE-mRNA stability. Unexpectedly, downregulation of all four AUF1 isoforms by targeting common exons did not affect fluorescence whereas selective downregulation of p40AUF1/p45AUF1 strongly increased fluorescence by stabilizing the GFP-ARE reporter mRNA. This observation was fully confirmed by the finding that only selective reduction of p40AUF1/p45AUF1 induced the production of GM-CSF, an endogenous target of AUF1. These data suggest that the relative levels of individual isoforms, rather than the absolute amount of AUF1, determine the net mRNA stability of ARE-containing transcripts, consistent with the differing ARE-binding capacities of the isoforms.
Asthma is characterized by bronchial inflammation and hyperresponsiveness that involves mast cell tryptase and potentially its specific receptor protease activated receptor 2 (PAR-2). Tryptase increases free intracellular calcium concentration ([Ca2+]i), a key step in activation of human airway smooth muscle cells (HASMC). The aim of this study was to analyze the effect of PAR-2 gene silencing on HASMC, in terms of calcium response, since no antagonist is available for this receptor. Five siRNA against PAR-2 were synthesized and transfected in HASMC using lipid agents, and PAR-2 expression was examined using Western blot, fluorescence-activated cell sorter, immunocytochemistry and RT-PCR. [Ca2+]i was measured using microspectrofluorimetry in response to tryptase, the activating peptide SLIGKV, trypsin, or caffeine. Two siRNA significantly inhibited PAR-2 expression in terms of both total and surface protein expression, as well as mRNA levels. Tryptase- and SLIGKV-induced transient increase in [Ca2+]i was significantly inhibited after transfection with the most appropriate siRNA, whereas neither trypsin nor caffeine response was altered. Two control siRNA had no effect in terms of both PAR-2 expression and calcium response. Transfection efficiency was maximal after 24 h and disappeared after 48 h. Gene silencing using siRNA can thus be used in vitro to assess the function of PAR-2 in HASMC.
In the present report, valosin-containing protein (VCP) was present in Mallory bodies (MBs). To determine if VCP plays a role in MB formation, primary cultured hepatocytes from drug-primed mice that spontaneously form MBs in vitro were studied. The results were compared with control normal hepatocytes. Gene-specific FITC-labeled gripNA (gVCP) was added to the medium of the primary cultures to inhibit the expression of VCP. gVCP increased MB formation by 230% in drug-primed mouse hepatocytes compared with primed liver cells where no VCP oligos were added. Blocking VCP expression induced both multiple small ubiquitin (Ub) and cytokeratin (CK) aggregates to form within the cytoplasm in normal mouse hepatocytes. Inhibition of VCP expression in both drug-primed and control hepatocytes caused a decrease in proteasome chymotrypsin-like (ChT-L) activity. Overexpression of VCP was achieved by transfecting the hepatocytes with a plasmid containing green fluorescent protein (GFP)-fused VCP (pVCP-GFP). Overexpressed VCP was located in both the cytoplasm and nucleus of pVCP-GFP overexpressing drug-primed hepatocytes. VCP was also concentrated within MBs. MB formation was not decreased by the overexpression of VCP in the cells. These results indicate that VCP plays an important role in inducing MB formation, probably through its molecular chaperone function in the ubiquitin-proteasome system (UPS).
The Src homology domain 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1) plays a key role in apoptosis and decreases phosphorylation of Akt. Apoptosis of cardiomyocytes is thought to contribute to the increased area of acute myocardial infarction (AMI), and Akt activation exerts a powerful cardioprotective effect after ischemia. Thus, a therapeutic strategy designed to inhibit expression of SHP-1 would be beneficial in AMI. Here we report that siRNA targeting SHP-1 reduced infarct size in a rat model of AMI. Upon injection into the ischemic left ventricular wall, the vector-based siRNA significantly suppressed the increase in the SHP-1 mRNA and the SHP-1 protein levels. The siRNA vector also significantly reduced the SHP-1 that bound to Fas-R. The SHP-1 siRNA vector increased phospho-Akt and reduced DNA fragmentation and caspase activity compared with the scramble siRNA vector. Finally, the area of myocardial infarction was significantly smaller with the SHP-1 siRNA vector than with the scramble siRNA vector at 2 days after LCA ligation. In conclusion, SHP-1 in the heart increased from the early stage of AMI, and this increase was thought to contribute to the increased area of myocardial infarction. Suppression of SHP-1 with the SHP-1 siRNA vector markedly reduced the infarct size in AMI.